Balanced chromosomal rearrangements associated with anomalous phenotypes are very valuable reagents used to identify candidate genes responsible for the phenotype and/or obtain molecular probes useful for further studies of other patients with similar phenotype. Furthermore, sequence analysis of the DNA flanking the breakpoints may provide insights as to the mechanisms leading to chromosome rearrangements, a major cause of birth defects. Here we propose the molecular characterization of the chromosomal breakpoints of an apparently balanced paracentric inversion associated with a multiple congenital anomaly-mental retardation syndrome. The phenotype of the patient resembles the Cornelia de Lange (CdL) phenotype in a relatively mild form. The breakpoints of the inversion are on the long arm of chromosome 3, bands 3q13 and 3q27. Interestingly, the chromosomal band 3q27 is definitely implicated in the Dup(3q) syndrome and, tentatively, in the CdL. The Dup (3q) syndrome is due to trisomy of a minimal segement in the 3q27 region also resembling the CdL phenotype. Preliminary mapping data led us to the identification of a cosmid clone spanning the 3q27 inversion breakpoint in our patient. Subclones from this cosmid identified junction fragments in genomic DNA from the patient. Random sequencing of cosmid subclones showed the presence of sequences identical to several ESTs deposited in public databases.